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Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
Reductively activated nitrous oxide reductase reacts directly with substrate
Jeannine M Chan1, John A Bollinger, Cassidy L Grewell
1Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, USA.
Journal of the American Chemical Society
|March 12, 2004
Summary
Nitrous oxide reductase converts N2O to N2 at its CuZ center. This study shows the activated enzyme binds N2O, revealing a potential catalytic intermediate state.
Area of Science:
- Biochemistry
- Microbiology
- Enzymology
Background:
- Bacterial denitrification converts nitrogen oxides, with nitrous oxide reductase (N2OR) catalyzing the final N2O to N2 conversion.
- The enzyme's active site, the mu4-sulfide bridged tetranuclear CuZ center, is crucial for this reaction.
Purpose of the Study:
- To investigate the substrate interaction and catalytic mechanism of N2OR from Achromobacter cycloclastes.
- To characterize the oxidation state of the CuZ center in the activated enzyme.
Main Methods:
- Isolation and characterization of reductively activated N2OR.
- Visible absorption and Electron Paramagnetic Resonance (EPR) spectroscopy.
- Enzyme assays with 15N-labeled N2O.
Main Results:
- Reductive activation by methyl viologen increased N2OR specific activity up to 15-fold.
- Spectroscopic analysis confirmed the CuZ center can reach a [4Cu(I)] oxidation state.
- Spectral changes upon N2O addition and detection of labeled product indicated substrate interaction and a potential catalytic intermediate.
Conclusions:
- The activated N2OR binds N2O, suggesting a substrate interaction step prior to steady-state turnover.
- A novel absorption band at 970 nm may signify a key catalytic intermediate in N2O reduction.
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